Immunoglobulin is a collective term for an antibody which recognizes invading foreign substances into the body of animal, etc. to trigger an immunoreaction, and a functionally and/or structurally similar polypeptide thereof, including types of IgG, IgM, IgA, IgD, IgE, and so on. In the fields of life science research, pharmaceuticals, and the laboratory testing, etc., there is an increasing demand for highly pure immunoglobulin.
Affinity chromatography is used as a core technology for a method of removing impurities to produce highly pure immunoglobulin.
In affinity chromatography, a polypeptide with high purity and high concentration can be purified through the following steps of (A)-(C):
(A) A step of loading a sample containing impurities into a column (loading step).
(B) A step of removing the impurities other than the polypeptide to be purified, from the loaded column (washing step).
(C) A step of collecting the polypeptide to be purified, from the column (elution step).
In that regard, in the loading and washing steps, it is essential that the environment inside the column is to set so that the polypeptide to be purified can tightly bind to an affinity ligand, whereas the internal environment of the column is changed in the elution step so that the both are dissociated, wherein the change in pH is usually used for this environmental change.
As a ligand for affinity chromatography used in purification of immunoglobulin, Protein A from Staphylococcus (hereafter referred to as Protein A) and its immunoglobulin binding domains which possess remarkably high specificity and affinity to the common region of immunoglobulin have been known and widely used in the manufacturing process of immunoglobulin in industrial scale.
However, there are problems resulting from the nature of immunoglobulin in the affinity chromatography where Protein A or a part thereof is used as a ligand, imposing limitations to the manufacture of immunoglobulin.
In order to carry out elution of immunoglobulin from a column by means of a pH change, a neutral pH range of 6-8 (pH used in the loading/washing steps), where the affinity between immunoglobulin and Protein A is high, needs to be changed to an acidic pH range of 3 to 4 (pH used in the elution step) where the affinity becomes very low. One of the problems is that final yield for the separation/purification of “immunoglobulin with native properties retained” may be significantly worsened in such an acidic range of pH since the change in the immunoglobulin conformation, i.e. association/aggregation may occur and compromise the functions (See Japanese Unexamined Patent Publication (Kokai) No. 2005-206602). In particular, humanized or human IgG, which is industrially most useful, e.g. used as monoclonal antibody drugs, has a higher affinity with Protein A than that of other immunoglobulins and thus requires a buffer with very strong acidity when eluted, leading to more probable association/aggregation. Therefore the inactivation of these IgGs has been a common problem in the art.
For example, supports used in industrial scale on which Protein A or its immunoglobulin binding domain is immobilized as an affinity ligand are available from GE healthcare biosciences, Millipore, etc. The manufacture of immunoglobulin using these supports also uses an acidic elution buffer (pH 3-4) upon elution.
Therefore, it is desired to develop a purification method which does not use acid pH in order to efficiently manufacture highly pure immunoglobulin.
Although various conventional techniques have been tried in order to solve the problem, they have the following problems and do not have much practical utility.
Methods by Adding Additives
The methods in which various kinds of additives are added to allow for elution at pH 5 to pH 7 in using Protein A, have problems such as low recovery ratios, unwanted effects due to the additives themselves, the necessity of removal of the additives with a charge (since they become an obstacle when ion exchange chromatography is used for industrial purification), remaining concern for damages on immunoglobulin at the elution pH of around 5, etc, and thus they have not been practical.
Methods Using of an Artificial Ligand Instead of Protein A
The methods using organic-chemically synthesized artificial ligands as alternatives of Protein A have disadvantages such as a) a significantly low enrichment factor due to slow elution, resulting in very inefficient purification, b) the necessity of concomitant use of additives (which is against the purpose of purification), and c) a poor ability to remove impurities.
Methods Using Shortened Time of Contact to an Acidic Solution
Although there are methods in which the contact time of antibody to an acidic solution is shortened, methods in which a high-concentration of neutral buffer is immediately mixed into an eluate, etc., these can not be a fundamental solution against association/aggregation of antibodies.
Methods Using a Mutant of Protein A as a Ligand
A method using a mutant of Protein A in which a hydrophobic amino acid in the immunoglobulin binding domain is replaced with a histidine to allow for elution at pH 5 (non-patent literature 1), leaves a concern for damages on immunoglobulin because it still uses acidic pH even though the pH is closer to neutral than that used for the naturally occurring version. In addition, the elution is too slow for an industrial process.
Methods in which the Three Dimensional Structure of Protein A is Destabilized (1)
On the other hand, losing the three dimensional structure of a polypeptide used as a ligand can be envisioned in order to disengage immunoglobulin from the ligand. The three dimensional structure of a polypeptide can be usually lost by adding a denaturant such as urea and guanidinium chloride, by increasing temperature, by removing a cofactor, or by changing salt concentration. In particular denaturants and temperature are effective for denaturing many polypeptides.
However, any of the above methods will cause the loss of the three dimensional structure of immunoglobulin to be purified, as well as that of Protein A or its domain, resulting in the irreversible loss of the biological functions of immunoglobulin.
Therefore, it is difficult to find a condition where the three dimensional structure of a ligand polypeptide alone is lost while immunoglobulin to be purified retains its three dimensional structure and its functions if the ligand polypeptide remains the wild type.
Methods in which the Three Dimensional Structure of Protein A is Destabilized (2)
In Z domain where a part of the amino acid sequence of B domain, one of the immunoglobulin binding domains of Protein A, is mutated, a mutant in which 6 glycines are inserted into the Loop 2 portion or a mutant in which the Loop 2 sequence is replaced by glycines became more unstable than Z domain in terms of the three dimensional structure. When this mutant is used, it is possible to elute immunoglobulin at pH 4.5 (non-patent literature 2). However, a concern for damages on immunoglobulin remains because it still uses acidic pH even though the pH is closer to neutral than the used for the naturally occurring version.
Methods in which the Three Dimensional Structure of Protein A is Destabilized (3)
It is known that Protein A is comprised of domains called E, D, A, B, and C domain each of which has about 60 amino acids, and these domains showing high homology each other can bind to the common region (Fc region) of immunoglobulin independently.
The mutant Z domain described above is derived from B domain (SEQ ID NO 1) by replacing Ala at position 1 with Val and Gly at position 29 with Ala in order to remove recognition sequences of polypeptide restriction enzymes. It is known that Z domain also binds to the common region (Fc region) of immunoglobulin either in a single domain configuration or in a multiple domain configuration.
However, the three dimensional structure of these B and Z domains is extremely stable, and even at pH 4 or lower and/or at a high temperature of 60° C. or above where immunoglobulin become unstable, their three dimensional structure is stable. Therefore, it is difficult to selectively destabilize only the three dimensional structure of these domains, which are ligands, by changing temperature without altering the three dimensional structure of immunoglobulin.
Each of immunoglobulin binding domains of said Protein A is comprised of the following (X)-(Z):
(X) Three helices (helix 1, helix 2, and helix 3 from the N-terminus)
(Y) Loop 1 which connects helix 1 and helix 2
(Z) Loop 2 which connects helix 2 and helix 3.
All of the amino acids directly involved in the binding with immunoglobulin are exposed to the surface of helix 1 and helix 2, and if these amino acids are replaced, then the binding specificities, etc. are likely to be changed.
On the other hand, it is known that the hydrophobic amino acids inside of the immunoglobulin binding domain do not directly make a contact with immunoglobulin and are not directly involved in binding specificity, but instead they do form hydrophobic bonding with other hydrophobic side chains which are spatially adjacent, significantly contributing to the stability of the three dimensional structure of the polypeptide.
It is supposed that the extent of the contribution generally correlates to a size of the side chain of a hydrophobic amino acid to some extent. It is also assumed that replacing an internal amino acid having a larger side chain with an amino acid having a smaller side chain can result in loss of hydrophobic bonding, and destabilize the native three dimensional structure of a polypeptide.
However, which position(s) of a hydrophobic amino acid(s) is(are) to be replaced with which amino acid(s) can not be easily determined in order to achieve the desired destabilization of the structure because the stability of the three dimensional structure of a polypeptide also depends on the characteristics of the individual polypeptide besides of the size(s) of the hydrophobic amino acid(s).
We have already investigated various replacements of hydrophobic amino acids having a small surface exposed area by amino acids having a smaller side chain in an immunoglobulin binding domain of Protein A, and revealed that mutants where a hydrophobic amino acid(s) around Loop 1 is replaced show the largest effects on the stability of the three dimensional structure of this domain (non-patent literature 3). However, this only suggests, to some extent, the correlation between an amino acid substitution and the stability of the three dimensional structure under a given condition of temperature (room temperature), and there is no description in relation to immunoglobulin.
On the other hand, if the three dimensional structure of Protein A is destabilized too much, a problem that its binding with immunoglobulin becomes difficult in the loading and washing steps, will arise in the first place. Therefore, the above studies could not be applicable to immunoglobulin purification as they are. That is, in order to purify immunoglobulin, it is required to be able to fully control the state where the three dimensional structure of a polypeptide for purification is stabilized, and the state where it is destabilized, by means of other than a pH change.
Note that a concept called the Gibbs free energy for denaturation is known as an indicator of the stability of the three dimensional structure of a polypeptide, and the descriptions about B domain of Protein A can be found in non-patent literatures 3 and 4.    [non-patent literature 1] Mol. Biotechnol. Vol. 10 (1998), P.9-16    [non-patent literature 2] J. Biotechnol. Vol. 76 (2000), P.233-244    [non-patent literature 3] Proc. Natl. Acad. Sci. USA Vol. 101 (2004), P.6952-6956    [non-patent literature 4] J. Mol. Biol. Vol. 372 (2007), P.254-267